Feedback-resistant pyruvate carboxylase gene from corynebacterium

ABSTRACT

The present invention relates to a mutated pyruvate carboxylase gene from  Corynebacterium . The mutant pyruvate carboxylase gene encodes a pyruvate carboxylase enzyme which is resistant to feedback inhibition from aspartic acid. The present invention also relates to a method of replacing the wild-type pyruvate carboxylase gene in  Corynebacterium  with this feedback-resistant pyruvate carboxylase gene. The present invention further relates to methods of the production of amino acids, preferably lysine, comprising the use of this mutant pyruvate carboxylase enzyme in microorganisms.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of the filing date of U.S.Provisional Application No. 60/239,913, filed on Oct. 13, 2000, which isincorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a mutated pyruvate carboxylase genefrom Corynebacterium. The mutant pyruvate carboxylase gene encodes apyruvate carboxylase enzyme which is resistant to feedback inhibitionfrom aspartic acid. The present invention also relates to a method ofreplacing the wild-type pyruvate carboxylase gene in Corynebacteriumwith this feedback-resistant pyruvate carboxylase gene. The presentinvention further relates to methods of the production of amino acids,preferably lysine, comprising the use of this mutant pyruvatecarboxylase enzyme in microorganisms.

2. Background Art

Pyruvate carboxylase is an important biotin-containing enzyme found in avariety of plants and animals, as well as some groups of bacteria(Modak, H. V. and Kelly, D. J., Microbiology 141:2619-2628 (1995)). Inthe presence of adenosine triphosphate (ATP) and magnesium ions,pyruvate carboxylase catalyzes the two-step carboxylation of pyruvate toform oxaloacetate, as shown in the equations below:

 ENZ−biotin−CŌ₂+Pyruvate→ENZ−biotin+oxaloacetate  (2)

In reaction (1) the ATP-dependent biotin carboxylase domain carboxylatesa biotin prosthetic group linked to a specific lysine residue in thebiotin-carboxyl-carrier protein (BCCP) domain. Acetyl-coenzyme Aactivates reaction (1) by increasing the rate of bicarbonate-dependentATP cleavage. In reaction (2), the BCCP domain donates the CO₂ topyruvate in a reaction catalyzed by the transcarboxylase domain(Attwood, P. V., Int. J. Biochem. Cell. Biol. 27:231-249 (1995)).

In bacteria such as Corynebacterium glutamicum, pyruvate carboxylase isutilized during carbohydrate metabolism to form oxaloacetate, which isin turn used in the biosynthesis of amino acids, particularly L-lysineand L-glutamate. Furthermore, in response to a cell's metabolic needsand internal environment, the activity of pyruvate carboxylase issubject to both positive and negative feedback mechanisms, where theenzyme is activated by acetyl-CoA, and inhibited by aspartic acid. Basedon its role in the pathway of amino acid synthesis, and its ability tobe regulated, pyruvate carboxylase plays a vital role in the synthesisof amino acids.

Bacteria such as C. glutamicum and E. coli are widely used in industryfor the production of amino acids such as L-glutamate and L-lysine.Because of the central importance of pyruvate carboxylase in theproduction of amino acids, particularly L-glutamate and L-lysine, theexploitation of pyruvate carboxylase to increase amino acid productionis of great interest in an industrial setting. Thus, promoting thepositive feedback mechanism of pyruvate carboxylase, or inhibiting itsnegative feedback mechanism, in C. glutamicum or could augment aminoacid production on an industrial scale.

BRIEF SUMMARY OF THE INVENTION

One aspect of the present invention relates to a nucleic acid moleculecomprising a nucleotide sequence which codes for a pyruvate carboxylaseof SEQ ID NO:19, wherein this pyruvate carboxylase contains at least onemutation which desensitizes the pyruvate carboxylase to feedbackinhibition by aspartic acid.

Another aspect of the present invention provides methods for using thenucleic acid of SEQ ID NO:1, which encodes the amino acid sequence of amutant pyruvate carboxylase. Such uses include the replacement of thewild-type pyruvate carboxylase with the feedback-resistant pyruvatecarboxylase, and the production of amino acids. An additional aspect ofthe present invention provides a polypeptide comprising the amino acidsequence of SEQ ID NO:2. Still another aspect of the present inventionprovides a polypeptide comprising the amino acid sequence selected fromthe group comprising SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ IDNO:12 SEQ ID NO:14, SEQ ID NO:16 and SEQ ID NO:18.

Another aspect of the present invention also relates to a nucleic acidmolecule comprising a nucleotide sequence which encodes the amino acidsequence of SEQ ID NO:2, or the amino acid sequence encoded by the DNAcontained in Deposit Number NRRL B-11474. Another aspect of the presentinvention further relates to a nucleic acid molecule comprising thenucleotide sequence of SEQ ID NO:1.

BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES

FIGS. 1A-1E show the full-length nucleotide sequence (SEQ ID NO:1)encoding the amino acid sequence of feedback-resistant pyruvatecarboxylase, and the corresponding amino acid sequence (SEQ ID NO:2).

FIG. 2 shows the amino acid sequences of the wild-type pyruvatecarboxylase (SEQ ID NO:19) isolated from Corynebacterium glutamicumATCC21253. The specific changes corresponding to the amino acid sequenceof the feedback-resistant pyruvate carboxylase (SEQ ID NO:2), isolatedfrom Corynebacterium glutamicum NRRL B-11474, are indicated.

FIG. 3 shows the effects of various substrate concentrations on thepyruvate carboxylase activity in C. glutamicum ATCC 21253 and NRRLB-11474.

FIG. 4 shows the effects of aspartate concentration on the activity ofpyruvate carboxylase in C. glutamicum ATCC21253 and NRRL B-11474.

FIG. 5 shows the effects of acetyl-CoA concentration on the activity ofpyruvate carboxylase in C. glutamicum ATCC21253 and NRRL B-11474.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to variations of the polypeptidecomprising the amino acid sequence which codes for the pyruvatecarboxylase as shown in SEQ ID NO:19. Preferably, the variations ofpyruvate carboxylase enzyme in the present invention contain at leastone mutation which desensitizes the pyruvate carboxylase to feedbackinhibition by aspartic acid. Such mutations may include deletions,insertions, inversions, repeats, and type substitutions. Morepreferably, the amino acid sequence mutation which desensitizes thewild-type pyruvate carboxylase enzyme (SEQ ID NO:19) to feedbackinhibition comprises at least one substitution selected from the groupconsisting of (a) methionine at position 1 being replaced with a valine,(b) glutamic acid at position 153 being replaced with an aspartic acid,(c) alanine at position 182 being replaced with a serine, (d) alanine atposition 206 being replaced with a serine, (e) histidine at position 227being replaced with an arginine, (f) alanine at position 455 beingreplaced with a glycine, and (g) aspartic acid at position 1120 beingreplaced with a glutamic acid. Still more preferably, the variation ofthe polypeptide encoded by the amino acid sequence of SEQ ID NO:19contains more than one of the above-mentioned mutations. Mostpreferably, the variation of the polypeptide encoded by the amino acidsequence of SEQ ID NO:19 contains all of the above-mentioned mutations.As one of ordinary skill in the art would appreciate, the numbering ofamino acid residues of a protein as used herein, begins at the aminoterminus (N-terminus) and proceeds towards the carboxy terminus(C-terminus), such that the first amino acid at the N-terminus isposition 1.

An embodiment of the present invention relates to an isolated orpurified nucleic acid molecule comprising a nucleotide sequence selectedfrom the group consisting of: (a) a nucleotide sequence which encodesthe amino acid sequence of SEQ ID NO:2 (b) a nucleotide sequenceencoding the amino acid sequence encoded by the DNA contained in DepositNumber NRRL B-11474 or (c) a nucleotide sequence complementary to any ofthe nucleotide sequences in (a) or (b).

Further embodiments of the invention include isolated nucleic acidmolecules that comprise a polynucleotide having a nucleotide sequence atleast 90% identical, and more preferably at least 95%, 97%, 98%, 99% or100% identical, to any of the nucleotide sequences in (a), (b), (c) or(d) above, or a polynucleotide which hybridizes under stringenthybridization conditions to a polynucleotide having a nucleotidesequence identical to a nucleotide sequence in (a), (b), (c) or (d)above. However, the polynucleotide which hybridizes does not hybridizeunder stringent hybridization conditions to a polynucleotide having anucleotide sequence consisting of only A residues or of only T residues.

Another aspect of the invention is directed to nucleic acid molecules atleast 90%, 95%, 97%, 98% or 99% identical to the nucleic acid sequenceshown in FIG. 1 (SEQ ID NO:1), or to the nucleic acid sequence of thedeposited DNA (NRRL B-30293, deposited May 12, 2000).

A further aspect of the invention provides a nucleic acid moleculecomprising a nucleotide sequence selected from the group consisting of:SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQID NO:15 and SEQ ID NO:17.

By a polynucleotide having a nucleotide sequence at least, for example,95% “identical” to a reference nucleotide sequence is intended that thenucleotide sequence of the polynucleotide is identical to the referencesequence except that the polynucleotide sequence may include up to fivepoint mutations per each 100 nucleotides of the reference nucleotidesequence encoding the pyruvate carboxylase polypeptide. In other words,to obtain a polynucleotide having a nucleotide sequence at least 95%identical to a reference nucleotide sequence, up to 5% of thenucleotides in the reference sequence may be deleted or substituted withanother nucleotide, or a number of nucleotides up to 5% of the totalnucleotides in the reference sequence may be inserted into the referencesequence.

As a practical matter, whether any particular nucleic acid molecule isat least 90%, 95%, 97%, 98% or 99% identical to, for instance, thenucleotide sequence shown in FIG. 1 or to the nucleotide sequence of thedeposited DNA can be determined conventionally using known computerprograms such as the FastA program. FastA performs a Pearson and Lipmansearch for similarity between a query sequence and a group of sequencesof the same type nucleic acid. Professor William Pearson of theUniversity of Virginia Department of Biochemistry wrote the FASTAprogram family (FastA, TFastA, FastX, TFastX and SSearch). Incollaboration with Dr. Pearson, the programs were modified anddocumented for distribution with GCG Version 6.1 by Mary Schultz and IrvEdelman, and for Versions 8 through 10 by Sue Olson.

Unless otherwise indicated, all nucleotide sequences determined bysequencing a DNA molecule herein were determined using an automated DNAsequencer (such as the ABI Prism 377). Therefore, as is known in the artfor any DNA sequence determined by this automated approach, anynucleotide sequence determined herein may contain some errors.Nucleotide sequences determined by automation are typically at leastabout 90% identical, more typically at least about 95% to at least about99.9% identical to the actual nucleotide sequence of the sequenced DNAmolecule.

Unless otherwise indicated, each “nucleotide sequence” set forth hereinis presented as a sequence of deoxyribonucleotides (abbreviated A, G, Cand T). However, by “nucleotide sequence” of a nucleic acid molecule orpolynucleotide is intended, for a DNA molecule or polynucleotide, asequence of deoxyribonucleotides, and for an RNA molecule orpolynucleotide, the corresponding sequence of ribonucleotides (A, G, Cand U) where each thymidine deoxynucleotide (T) in the specifieddeoxynucleotide sequence is replaced by the ribonucleotide uridine (U).For instance, reference to an RNA molecule having the sequence of SEQ IDNO:1 set forth using deoxyribonucleotide abbreviations is intended toindicate an RNA molecule having a sequence in which each deoxynucleotideA, G or C of SEQ ID NO:1 has been replaced by the correspondingribonucleotide A, G or C, and each deoxynucleotide T has been replacedby a ribonucleotide U.

As indicated, nucleic acid molecules of the present invention may be inthe form of RNA, such as mRNA, or in the form of DNA, including, forinstance, DNA and genomic DNA obtained by cloning or producedsynthetically. The DNA may be double-stranded or single-stranded.Single-stranded DNA or RNA may be the coding strand, also known as thesense strand, or it may be the non-coding strand, also referred to asthe anti-sense strand.

By “isolated” nucleic acid molecule(s) is intended a nucleic acidmolecule, DNA or RNA, which has been removed from its nativeenvironment. For example, recombinant DNA molecules contained in avector are considered isolated for the purposes of the presentinvention. Further examples of isolated DNA molecules includerecombinant DNA molecules maintained in heterologous host cells orpurified (partially or substantially) DNA molecules in solution.Isolated RNA molecules include in vivo or in vitro RNA transcripts ofthe DNA molecules of the present invention. Isolated nucleic acidmolecules according to the present invention further include suchmolecules produced synthetically.

In another aspect, the invention provides an isolated nucleic acidmolecule comprising a polynucleotide which hybridizes under stringenthybridization conditions to a portion of the polynucleotide in a nucleicacid molecule of the invention described herein. By “stringenthybridization conditions” is intended overnight incubation at 42° C. ina solution comprising: 50% formamide, 5× SSC (150 mM NaCl, 15 mMtrisodium citrate), 50 mM sodium phosphate (pH 7.6), 5× Denhardt'ssolution, 10% dextran sulfate, and 20 μg/ml denatured, sheared salmonsperm DNA, followed by washing the filters in 0.1× SSC at about 65° C.By a polynucleotide which hybridizes to a “portion” of a polynucleotideis intended a polynucleotide (either DNA or RNA) hybridizing to at leastabout 15 nucleotides (nt), and more preferably at least about 20 nt,still more preferably at least about 30 nt, and even more preferablyabout 30-70 nt of the reference polynucleotide. These are useful asdiagnostic probes and primers.

Of course, polynucleotides hybridizing to a larger portion of thereference polynucleotide (e.g., the deposited plasmid), for instance, aportion 25-750 nt in length, or even to the entire length of thereference polynucleotide, are also useful as probes according to thepresent invention, as are polynucleotides corresponding to most, if notall, of the nucleotide sequences of any of the nucleotide sequencesincluded in the present intention. By a portion of a polynucleotide of“at least 20 nt in length,” for example, is intended 20 or morecontiguous nucleotides from any of the nucleotide sequences of thereference polynucleotides, (e.g., the deposited DNA or the nucleotidesequence as shown in any of the figures). As indicated, such portionsare useful diagnostically either as a probe, according to conventionalDNA hybridization techniques, or as primers for amplification of atarget sequence by the polymerase chain reaction (PCR), as described,for instance, in Molecular Cloning, A Laboratory Manual, 2nd. edition,edited by Sambrook, J., Fritsch, E. F. and Maniatis, T., (1989), ColdSpring Harbor Laboratory Press, the entire disclosure of which is herebyincorporated herein by reference.

The nucleic acid molecules of the present invention are suitable for usein vectors. As such, polynucleotides of interest can be joined to thenucleic acid molecules of the present invention, which may optionallycontain selectable markers. A preferred embodiment of the presentinvention is that the vector comprises a functional Corynebacteriumreplication origin. A replication origin is a nucleotide sequence,typically several hundred base pairs long, that is vital to theinitiation of DNA replication.

The vectors can optionally contain an exogenous terminator oftranscription; an exogenous promoter; and a discrete series ofrestriction endonuclease recognition sites, said series being betweensaid promoter and said terminator. The vector can optionally containtheir native expression vectors and/or expression vectors which includechromosomal-, and episomal-derived vectors, e.g., vectors derived frombacterial exogenous plasmids, bacteriophage, and vectors derived fromcombinations thereof, such as cosmids and phagemids.

A DNA insert of interest should be operatively linked to an appropriatepromoter, such as its native promoter or a host-derived promoter, thephage lambda P_(L) promoter, the phage lambda P_(R) promoter, the E.coli lac promoters, such as the lacI and lacZ promoters, trp and tacpromoters, the T3 and T7 promoters and the gpt promoter to name a few.Other suitable promoters will be known to the skilled artisan.

The expression constructs will further contain sites for transcriptioninitiation, termination and, in the transcribed region, a ribosomebinding site for translation. The coding portion of the maturetranscripts expressed by the constructs can include a translationinitiating codon at the beginning and a termination codon appropriatelypositioned at the end of the polypeptide to be translated.

As indicated, the expression vectors will preferably include at leastone selectable marker. Preferably the selection marker comprises anucleotide sequence which confers antibiotic resistance in a host cellpopulation. Such markers include amikacin, augmentin (amoxicillin plusclavulonic acid), ampicillin, cefazolin, cefoxitin, ceftazidime,ceftiofur, cephalothin, enrofloxacin, florfenicol, gentamicin, imipenem,kanamycin, penicillin, sarafloxicin, spectinomycin, streptomycin,tetracycline, ticarcillin, tilmicosin, or chloramphenicol resistancegenes. Other suitable markers will be readily apparent to the skilledartisan.

The invention also provides for a method of producing a host cell wherethe expression vectors of the current invention have been introducedinto the host cell. Methods of introducing genetic material into hostcells, such as those described in typical molecular biology laboratorymanuals, for example J. Sambrook, E. F. Fritsch and T. Maniatis,Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1989), are well known to theskilled artisan. These methods include, but are not limited to, calciumphosphate transfection, DEAE-dextran mediated transfection,microinjection, lipid-mediated transfection, electroporation orinfection. Accordingly, a preferred embodiment of the present inventionprovides a host cell comprising the vector of the present invention.

As used in the present invention, a host cell refers to any prokaryoticor eukaryotic cell where the desired nucleic acid sequence has beenintroduced into the cell. There are a variety of suitable host cells,including but not limited to bacterial, fungal, insect, mammalian andplant cells, that can be utilized in the present invention.Representative bacterial host cells include, but are not limited to,Streptococci, Staphylococci, E. coli, Streptomyces, Bacillus andCorynebacterium. Representative fungal cells include but are not limitedto, yeast cells and Aspergillus. Insect cells include, but are notlimited to, Drosophila S2 and Spodoptera Sf9 cells. Examples ofmammalian cells include, but are not limited to, CHO, COS and Helacells.

The present invention provides methods for utilizing the nucleic acid ofSEQ ID NO:1, which encodes the amino acid sequence of a mutant pyruvatecarboxylase. Such methods include the replacement of the wild-typepyruvate carboxylase with the feedback-resistant pyruvate carboxylase,and the production of amino acids. The method for replacement of awild-type pyruvate carboxylase gene, with a feedback resistant pyruvatecarboxylase gene, in a Corynebacterium glutamicum host cell comprisesthe steps of: (a) replacing a genomic copy of the wild-type pyruvatecarboxylase gene with a selectable marker gene through homologousrecombination to form a first recombinant strain; and (b) replacing theselectable marker gene of step (a) in the first recombinant strain, withthe feedback resistant pyruvate carboxylase gene through homologousrecombination to form a second recombinant strain. The homologousrecombination in steps (a) and (b) would occur between the geneticmaterial of the host cell and any of the vectors of the presentinvention.

Homologous recombination is a technique that is used to disruptendogenous nucleotide sequences in a host cell. Normally, when anexogenous nucleotide sequence is inserted into a host cell, thispolynucleotide may randomly insert into any area of the host cell'sgenome, including endogenous plasmids. However, with homologousrecombination, the exogenous nucleotide sequence contains sequences thatare homologous to an endogenous nucleotide sequence within the hostcell. Once introduced into the cell, for example by electroporation, theexogenous nucleotide sequence will preferentially recombine with andreplace the endogenous nucleotide sequence with which it is homologous.

As used herein, an exogenous nucleotide sequence, is a nucleotidesequence which is not found in the host cell. Thus, the term exogenousnucleotide sequence is meant to encompass a nucleotide sequence that isforeign to the host cell, as well as a nucleotide sequence endogenous,or native, to the host cell that has been modified. Modification of theendogenous nucleotide sequence may include, for instance, mutation ofthe native nucleotide sequence or any of its regulatory elements. Asused herein, mutation is defined as any change in the wild-type sequenceof the host's genetic material, including plasmid DNA. An additionalform of modification may also include fusion of the endogenousnucleotide sequence to a nucleotide sequence that is normally notpresent, in relation to the endogenous nucleotide sequence.

Host cells that have undergone homologous recombination are selected onthe basis of antibiotic resistance through the use of, for example, theselectable markers mentioned above. The process of selecting cells thathave undergone homologous recombination will be readily apparent to oneskilled in the art.

Another aspect of the current invention is a method for producing aminoacids. In the current context, production of amino acids is accomplishedby culturing host cells where a vector of the present invention has beenintroduced into the host cell, or culturing host cells where homologousrecombination, involving a vector of the present invention, has takenplace. Culturing of the host cells is performed in the appropriateculture media. Subsequent to culturing the host cells in culture media,the desired amino acids are separated from the culture media.Preferably, the amino acids produced by the methods described hereininclude L-lysine, L-threonine, L-methionine, L-isoleucine, L-glutamate,L-arginine and L-proline. More preferably, the present invention relatesto the production of L-lysine.

The present invention provides an isolated or purified polypeptideencoded by the DNA plasmid encoding pyruvate carboxylase contained inDeposit Number NRRL B-30293, or the amino acid sequence of SEQ ID NO:2.Still another aspect of the present invention provides a polypeptidecomprising the amino acid sequence selected from the group consisting ofSEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQID NO:16 and SEQ ID NO:18.

Accordingly, SEQ ID NO:6 corresponds to the amino acid sequence:PSKNIDDIVKSAE. SEQ IN NO:8 corresponds to the amino acid sequence:RGMRFVSSPDELR. SEQ ID NO:10 corresponds to the amino acid sequence:AAFGDGSVYVEFA. SEQ ID NO:12 corresponds to the amino acid sequence:VQILGDRTGEVVH. SEQ ID NO:14 corresponds to the amino acid sequence:IATGFIGDHPHLL. SEQ ID NO:16 corresponds to the amino acid sequence:TITASVEGKIDRV. SEQ ID NO:18 corresponds to the amino acid sequence:MTAITLGGLLLKGIITLV.

All of the polypeptides of the present invention are preferably providedin an isolated form. As used herein, “isolated polypeptide” is intendedto mean a polypeptide removed from its native environment. Thus, apolypeptide produced and/or contained within a recombinant host cell isconsidered isolated for purposes of the present invention. Also intendedas an “isolated polypeptide” are polypeptides that have been purified,partially or substantially, from a recombinant host. For example, arecombinantly produced version of the pyruvate carboxylase enzyme can besubstantially purified by the one-step method described in Smith andJohnson, Gene 67:31-40 (1988).

One aspect of the present invention include the polypeptides which areat least 80% identical, more preferably at least 90%, 95% or 100%identical to the polypeptide encoded by the DNA plasmid encodingpyruvate carboxylase contained in Deposit Number NRRL B-30293, thepolypeptide of SEQ ID NO:2.

By a polypeptide having an amino acid sequence at least, for example,95% “identical” to the amino acid sequence of SEQ ID NO:2, for example,it is intended that the amino acid sequence of the polypeptide isidentical to the reference sequence except that the polypeptide sequencemay include up to five amino acid alterations per each 100 amino acidsof the amino acid sequence of SEQ ID NO:2, for example. In other words,to obtain a polypeptide having an amino acid sequence at least 95%identical to a reference amino acid sequence, up to 5% of the amino acidresidues in the reference sequence may be deleted or substituted withanother amino acid, or a number of amino acids up to 5% of the totalamino acid residues in the reference sequence may be inserted into thereference sequence. These alterations of the reference sequence mayoccur at the amino or carboxy terminal positions of the reference aminoacid sequence or anywhere between those terminal positions, interspersedeither individually among residues in the reference sequence or in oneor more contiguous groups within the reference sequence.

As a practical matter, whether any particular polypeptide is, forinstance, 95% identical to the amino acid sequence shown in SEQ ID NO:2,or to the amino acid sequence encoded by deposited DNA clone can bedetermined conventionally using known computer programs such the Bestfitprogram (Wisconsin Sequence Analysis Package, Version 8 for Unix,Genetics Computer Group, University Research Park, 575 Science Drive,Madison, Wis. 53711). When using Bestfit or any other sequence alignmentprogram to determine whether a particular sequence is, for instance, 95%identical to a reference sequence according to the present invention,the parameters are set, of course, such that the percentage of identityis calculated over the full length of the reference amino acid sequenceand that gaps in homology of up to 5% of the total number of amino acidresidues in the reference sequence are allowed.

Another aspect of the present invention provides a nucleic acid moleculeencoding the polypeptide comprising the amino acid sequence selectedfrom the group consisting of SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQID NO:12, SEQ ID NO:14, SEQ ID NO:16 and SEQ ID NO:18. Preferably, theinvention provides for nucleic acid molecules, which code for theaforementioned polypeptides, that are selected from the group consistingof SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13,SEQ ID NO:15 and SEQ ID NO:17.

Accordingly, SEQ ID NO:5 corresponds to the nucleic acid sequence thatcodes for the amino acid sequence of SEQ ID NO:6. SEQ ID NO:7corresponds to the nucleic acid sequence that codes for the amino acidsequence of SEQ ID NO:8. SEQ ID NO:9 corresponds to the nucleic acidsequence that codes for the amino acid sequence of SEQ ID NO:10. SEQ IDNO:11 corresponds to the nucleic acid sequence that codes for the aminoacid sequence of SEQ ID NO:12. SEQ ID NO:13 corresponds to the nucleicacid sequence that codes for the amino acid sequence of SEQ ID NO:14.SEQ ID NO:15 corresponds to the nucleic acid sequence that codes for theamino acid sequence of SEQ ID NO:16. SEQ ID NO:17 corresponds to thenucleic acid sequence that codes for the amino acid sequence of SEQ IDNO:18.

Methods used and described herein are well known in the art and are moreparticularly described, for example, in J. H. Miller, Experiments inMolecular Genetics, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. (1972); J. H. Miller, A Short Course in Bacterial Genetics,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1992); M.Singer and P. Berg, Genes & Genomes, University Science Books, MillValley, Calif. (1991); J. Sambrook, E. F. Fritsch and T. Maniatis,Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1989); P. B. Kaufman et al.,Handbook of Molecular and Cellular Methods in Biology and Medicine, CRCPress, Boca Raton, Fla. (1995); Methods in Plant Molecular Biology andBiotechnology, B. R. Glick and J. E. Thompson, eds., CRC Press, BocaRaton, Fla. (1993); P. F. Smith-Keary, Molecular Genetics of Escherichiacoli, The Guilford Press, New York, N.Y. (1989); Plasmids: A PracticalApproach, 2nd Edition, Hardy, K. D., ed., Oxford University Press, NewYork, N.Y. (1993); Vectors: Essential Data, Gacesa, P., and Ramji, D.P., eds., John Wiley & Sons Pub., New York, N.Y. (1994); Guide toElectroporation and electrofusions, Chang, D., et al., eds., AcademicPress, San Diego, Calif. (1992); Promiscuous Plasmids of Gram-NegativeBacteria, Thomas, C. M., ed., Academic Press, London (1989); The Biologyof Plasmids, Summers, D. K., Blackwell Science, Cambridge, Mass. (1996);Understanding DNA and Gene Cloning: A Guide for the Curious, Drlica, K.,ed., John Wiley and Sons Pub., New York, N.Y. (1997); Vectors: A Surveyof Molecular Cloning Vectors and Their Uses, Rodriguez, R. L., et al.,eds., Butterworth, Boston, Mass. (1988); Bacterial Conjugation, Clewell,D. B., ed., Plenum Press, New York, N.Y. (1993); Del Solar, G., et al.,Replication and control of circular bacterial plasmids,” Microbiol. Mol.Biol. Rev. 62:434-464(1998); Meijer, W. J., et al., “Rolling-circleplasmids from Bacillus subtilis: complete nucleotide sequences andanalyses of genes of pTA1015, pTA1040, pTA1050 and pTA1060, andcomparisons with related plasmids from gram-positive bacteria,” FEMSMicrobiol. Rev. 21:337-368 (1998); Khan, S. A., “Rolling-circlereplication of bacterial plasmids,” Microbiol. Mol. Biol. Rev.61:442-455 (1997); Baker, R. L., “Protein expression using ubiquitinfusion and cleavage,” Curr. Opin. Biotechnol. 7:541-546 (1996);Makrides, S. C., “Strategies for achieving high-level expression ofgenes in Escherichia coli,” Microbiol. Rev. 60:512-538 (1996); Alonso,J. C., et al, “Site-specific recombination in gram-positivetheta-replicating plasmids,” FEMS Microbiol. Lett. 142:1-10 (1996);Miroux, B., et al., “Over-production of protein in Escherichia coli:mutant hosts that allow synthesis of some membrane protein and globularprotein at high levels,” J. Mol. Biol. 260:289-298 (1996); Kurland, C.G., and Dong, H., “Bacterial growth inhibited by overproduction ofprotein,” Mol. Microbiol. 21:1-4 (1996); Saki, H., and Komano, T., “DNAreplication of IncQ broad-host-range plasmids in gram-negativebacteria,” Biosci. Biotechnol. Biochem. 60:377-382 (1996); Deb, J. K.,and Nath, N., “Plasmids of corynebacteria,” FEMS Microbiol. Lett.175:11-20 (1999); Smith, G. P., “Filamentous phages as cloning vectors,”Biotechnol. 10:61-83 (1988); Espinosa, M., et al., “Plasmid rollingcicle replication and its control,” FEMS Microbiol. Lett. 130:111-120(1995); Lanka, E., and Wilkins, B. M., “DNA processing reaction inbacterial conjugation,” Ann. Rev. Biochem. 64:141-169 (1995);Dreiseikelmann, B., “Translocation of DNA across bacterial membranes,”Microbiol. Rev. 58:293-316 (1994); Nordstrom, K., and Wagner, E. G.,“Kinetic aspects of control of plasmid replication by antisense RNA,”Trends Biochem. Sci. 19:294-300 (1994); Frost, L. S., et al., “Analysisof the sequence gene products of the transfer region of the F sexfactor,” Microbiol. Rev. 58:162-210 (1994); Drury, L., “Transformationof bacteria by electroporation,” Methods Mol. Biol. 58:249-256 (1996);Dower, W. J., “Electroporation of bacteria: a general approach togenetic transformation,” Genet. Eng. 12:275-295 (1990); Na, S., et al.,“The factors affecting transformation efficiency of coryneform bacteriaby electroporation,” Chin. J. Biotechnol. 11:193-198 (1995); Pansegrau,W., “Covalent association of the traI gene product of plasmid RP4 withthe 5′-terminal nucleotide at the relaxation nick site,” J. Biol. Chem.265:10637-10644 (1990); and Bailey, J. E., “Host-vector interactions inEscherichia coli,” Adv. Biochem. Eng. Biotechnol. 48:29-52 (1993).

EXAMPLES

The following examples are illustrative only and are not intended tolimit the scope of the invention as defined by the appended claims.

Strains and Media

Bacterial strains used were Corynebacterium glutamicum ATCC 21253 andNRRL B-11474. These strains have an auxotrophy for homoserine (ATCC21253) and for threonine, methionine and alanine (NRRL B-1 1474).

Defined medium for Corynebacterium glutamicum ATCC 21253 contained thefollowing ingredients (per liter): glucose, 20 g; NaCl, 2 g; citrate(trisodium salt, dihydrate), 3 g; CaCl₂.2H₂O, 0.1 g; MgSO₄.7H₂O, 0.5 g;Na₂EDTA.2H₂O, 75 mg; FeSO₄.7H₂O, 50 mg; 100× salt solution, 20 ml;K₂HPO₄, 4 g; KH₂PO₄, 2 g; (NH₄)₂SO₄, 7.5 g; urea, 3.75 g; leucine, 0.1g; threonine, 0.15 g; methionine, 0.05 g; thiamine, 0.45 mg; biotin,0.45 mg; pantothenic acid, 4.5 mg (pH 7.0). The salt solution containedthe following ingredients (per liter): MnSO₄, 200 mg; Na₂B₄O₇.10H₂O, 20mg; (NH₄)₆Mo₇O₂₄.4H₂O, 10 mg; FeCl₃.6H₂O, 200 mg; ZnSO₄.7H₂O, 50 mg;CuCl₂.2H₂O, 20 mg (pH 2.0).

Defined medium for Corynebacterium glutamicum NRRL B-1 1474 containedthe following ingredients (per liter): glucose, 20 g; NaCl, 1 g,MgSO₄.7H₂O, 0.4 g; FeSO₄.7H₂O, 0.01 g; MnSO₄.H₂O, 0.01 g; KH₂PO₄, 1 g;(NH₄)₂SO₄, 10 g; urea, 2.5 g; alanine, 0.5 g; threonine, 0.25 g;methionine, 0.5 g; thiamine, 0.45 mg; biotin, 0.45 mg; niacinamide, 50mg (pH 7.2).

Pyruvate Carboxylase and Phosphoenol Pyruvate Carboxylase Assay

Pyruvate carboxylate and phosphoenol pyruvate carboxylate assays wereperformed with permeabilized cells prepared by the following method. Logphase cells were harvested by centrifugation for 10 min at 5000×g at 4°C. and washed with 20 ml of the ice-cold washing buffer (50 mM Tris/HCl[pH 6.3] containing 50 mM NaCl). The cell pellet was resuspended in anice-cold Hepes buffer (100 mM Hepes [pH 7.5] containing 20% Glycerol) toreach a final concentration of 25 g dry cell weight/liter. Resuspendedcells were permeabilized by adding 30 μl of a 10%Hexadecyltrimethyl-ammonium bromide (CTAB) (w/v) solution to 1 ml ofcells to give a final concentration of 0.3% (CTAB)(v/v).

For determination of pyruvate carboxylate activity, the assay mixturecontained 10 mM pyruvic acid, 14 mM KHCO₃, 4 mM MgCl₂, 1.75 mM ATP, 50μmole acetyl-CoA, 0.3 mg bovine serum albumin, 0.055 U citrate synthaseand 50 mM sodium phosphate buffer ([pH 7.5] containing 0.1 mg5,5′-Dithio-bis(2-nitrobenzoic acid) (DTNB)) in a final volume of 1 ml.The reaction was started at 30° C. with the addition of 10 μl of thepermeabilized cell suspension, and the formation of DTNB-thiophenolatewas followed over time at 412 nm. Relevant standards and controls werecarried out in the same manner.

For determination of phosphoenol pyruvate carboxylase activity, theassay mixture contained 10 mM phosphoenol pyruvate, 14 mM KHCO₃, 4 mMMgCl₂, 50 μmole acetyl-CoA, 0.3 mg bovine serum albumin, 0.055 U citratesynthase and 50 mM sodium phosphate buffer ([pH 7.5] containing 0.1 mg5,5′-Dithio-bis(2-nitrobenzoic acid) (DTNB)) in a final volume of 1 ml.The reaction was carried out in the same conditions described for thepyruvate carboxylase assay.

The reproducibility for enzyme assays was typically 10%.

DNA Isolation and Purification

DNA was isolated from cultures of NRRL B-11474 cells. Defined media forNRRL B-11474 (CM media) contain the following ingredients, per liter:sucrose, 50 g; KH₂PO₄, 0.5 g; K₂HPO₄, 1.5 g; urea, 3 g; MgSO₄.7H₂O, 0.5g; polypeptone, 20 g; beef extract, 5 g; biotin, 12.5 ml (60 mg/L);thiamine, 25 ml (120 mg/L), niacinamide, 25 ml (5g/L); L-methionine, 0.5g; L-threonine, 0.25 g; L-alanine, 0.5 g. NRRL B-11474 cells wereharvested from CM media and suspended in 10 ml of TE, pH 8 (10 mMTris*Cl, 1 mM EDTA). Forty micrograms of RNase A and 10 milligrams oflysozyme were added per milliliter of suspension and the suspension wasincubated at 37° C. for 30 minutes. The suspension was made in 1.0% insodiumdodecyl sulfate (SDS) and 0.1 mg/l proteinase K was added, and thecells were lysed by incubation at 37° C. for 10 minutes. Nucleic acidswere purified by three extractions with TE-saturated phenol (pH 7),followed by ethanol precipitation. Nucleic acid precipitates were twicewashed with 80% ethanol and redissolved in TE pH 8.

The concentrations of DNA were quantified spectrophotometrically at 260nm. Purity of DNA preparations were determined spectrophotometrically(A260/A280 and A26JA230 ratios) and by agarose gel electrophoresis (0.8%agarose in 1× TAE).

Sequencing of the genomic DNA was performed, as is known by one ofordinary skill in the art, by creating libraries of plasmids and cosmidsusing pGEM3 and Lorist 6 respectively. Briefly, a Sau3AI digestion wasperformed on the genomic DNA and inserted into the BamHI site of pGEM3.The forward primer was used to generate a sequence, and primer walkinggenerated the remainder of the sequence.

Activity of Pyruvate Carboxylase

Development of a Continuous Assay for Determining Pyruvate CarboxylaseActivity

A discontinuous assay for determining pyruvate carboxylase frompermeabilized cells has been previously described (Peters-Wendisch, P.G. et al. Microbiology, 143: 1095-1103 (1997)). Because of the centrallocation of OAA in the metabolism, it seemed to be that OAA wouldaccumulate during the first reaction of the discontinuous assay. Mostlikely, OAA would be lost to other products, because of the competingenzymes that are still active. This depletion of OAA would inevitablylead to the underestimation of pyruvate carboxylase activity. To verifythis assumption of decreasing OAA concentrations, a known amount of OAAwas added to the first reaction in presence of permeabilized andnon-permeabilized cells. A significant loss of OAA was detected,demonstrating that permeabilized cells are capable of furthertransformation of OAA.

To account for the intrinsic loss of OAA during the experiment, acontinuous assay was carried out by coupling the two-reaction assay to aone-reaction assay in presence of an excess of citrate synthase. Theamount of permeabilized cells added in the assay was optimized to obtaina detectable activity, with the lowest possible background absorbencydue to the presence of cells.

To confirm that the continuous assay specifically detected pyruvatecarboxylase activity, controls were carried out by assaying for activityin absence of each reaction component (Table 1). Using these controls,the detected activity was determined to be a carboxylation reactionrequiring pyruvate, Mg and ATP.

TABLE 1 Controls for the continuous pyruvate carboxylate assay. DetectedActivity Control (Abs/min.mg DCW) Complete mixture 0.30 Cells omitted 0Pyruvate omitted 0.01 KHCO₃ omitted 0.03 MgCl₂ omitted 0.02 ATP omitted0.03 Citrate synthase omitted 0.10 Complete + biotin 0.35 Complete +avidin Not determined yetTo optimize the assay, the influence of the ratio of CTAB:cells wastested. Maximal activity was measured between 8 and 24 mg CTAB/mg drycell weight (DCW). Pyruvate carboxylase activity was measured in cellsincubated with CTAB with varying incubation times. The activity ofpyruvate carboxylase remained constant within 0 and 5 minutes.Similarly, different concentrations of DTNB, within the range 0.1-0.3g/l, gave identical pyruvate carboxylase activity. To confirm theability of the assay for determining pyruvate carboxylase activity inCorynebacterium glutamicum, different quantities of cells were used.Linearity between enzyme activity and quantity of cells was observedwithin the range 0-0.3 mg DCW.Enzymology Study of Pyruvate Carboxylase from Corynebacteriumglutamicum: Behavior of Pyruvate Carboxylase Towards Its Substrates

Pyruvate carboxylase activity was determined as a function of variousconcentrations of its substrates: pyruvate, bicarbonate and ATP (FIG.3). Based on the data generated, the affinity constants of pyruvatecarboxylase for its substrates were determined (Table 2). The pyruvatecarboxylase from NRRL B-11474 (also known as BF100) and ATCC 21253strains demonstrated a similar affinity for pyruvate and ATP. Pyruvatecarboxylase activity in both strains were inhibited by ATP above aconcentration of 2 mM. However pyruvate carboxylase in ATCC 21253 had ahigher affinity for bicarbonate than pyruvate carboxylase from NRRLB-11474 (BF100).

TABLE 2 Comparison of affinity constants for substrates on pyruvatecarboxylate from C. glutamicum, BF100 and ATCC 21253. StrainK_(M(pyruvate))[mM] K_(M(HCO) ₃ ⁻ ₎[mM] K_(M(ATP))[mM] C. glutamicum Pyc1.3 ± 0.3 14.4 ± 4   0.4 ± 0.1 Pyc ATCC 21253 0.3 ± 0.1 2.9 ± 0.8 0.3 ±0.1Table 2: Comparison of affinity constants for substrates on pyruvatecarboxylate from C. glutamicum BF100 and ATCC 21253.Aspartate Inhibition of Pyruvate Carboxylase

Aspartate inhibits phosphoenol pyruvate carboxylase (PEPC) activity. Todetermine the effect of aspartate on the activity of pyruvatecarboxylase, aspartate was added at different concentrations in thespectrophotometer cuvette and enzyme activities were measured. As acomparison, the same experiment was carried out with PEPC in ATCC 21253(FIG. 5).

The PEPC of Corynebacterium glutamicum (ATCC 21253) was found to bestrongly inhibited by aspartate. The enzyme was completely inhibitedwith a concentration of 5 mM aspartate. However, pyruvate carboxylasefrom the same strain was less sensitive to aspartate, i.e. it retained35% of its original activity in the presence of 25 mM aspartate.

The pyruvate carboxylase activity in NRRL B-11474 showed a higher basalpyruvate carboxylase activity than ATCC 21253, i.e. the pyruvatecarboxylase activity was about 5-times higher in NRRL B-11474 than inthe ATCC 21253. Moreover, a dramatic difference in their aspartateinhibition patterns was found. Pyruvate carboxylase from NRRL B-11474strain was activated by low aspartate concentrations within the range0-30 mM and inhibited within the range 30-100 mM aspartate. Neverthelessit retained 50% of its original activity, even in the presence of 100 mMaspartate. Activity was maintained at 30% in the presence of 500 mMaspartate. On the other hand, Pyruvate carboxylase from ATCC 21253 wasfound to be more sensitive to aspartate than pyruvate carboxylase fromNRRL B-11474. The pyruvate carboxylase from ATCC 21253 lost 70% of itsoriginal activity at a concentration of 30 mM aspartate.

Activation of Pyruvate Carboxylase by Acetyl-CoA

The feedback resistant pyruvate carboxylase gene of the presentinvention was isolated and cloned from NRRL B-11474. The isolated/clonedpyruvate carboxylase gene has been deposited in an E. coli host cell;lunder deposit NRRL B-30923. Deposit Number NRRL B-30293 was deposited onMay 12, 2000 the Agricultural Research Culture Collection (NRRL),International Depository Authority; 1815 North University Street;Peoria, Ill., 651064 U.S.A. All strains were deposited under the termsof the Budapest Treaty.

Pyruvate carboxylase activity was measured in the presence of differentconcentrations of acetyl-CoA (FIG. 6). Pyruvate carboxylase activity inboth strains increased with increasing acetyl-CoA concentrations. Theeffect of acetyl-CoA on citrate synthase itself was studied also.Acetyl-CoA had a Km of 10 μM, demonstrating that under our conditions,citrate synthase is saturated with acetyl-CoA. Therefore, the increasingactivity of pyruvate carboxylase with increasing acetyl-CoAconcentration is the result of acetyl-CoA activating pyruvatecarboxylase.

All publications mentioned herein above are hereby incorporated in theirentirety by reference.

While the foregoing invention has been described in some detail forpurposes of clarity and understanding, it will be appreciated by oneskilled in the art from a reading of this disclosure that variouschanges in form and detail can be made without departing from the truescope of the invention and appended claims.

1. An isolated or purified nucleic acid molecule comprising a nucleotidesequence selected from the group consisting of: a) the nucleotidesequence encoding amino acids 1 to 1157 of SEQ ID NO:2; b) a nucleotidesequence encoding the amino acid sequence encoded by the DNA plasmidencoding feedback resistant pyruvate carboxylase enzyme, said plasmidcontained in Deposit Number NRRL B-30293; and c) a nucleotide sequencecompletely complementary to any of the nucleotide sequences (a) or (b).2. The nucleic acid molecule of claim 1, comprising the nucleotidesequence of SEQ ID NO:1.
 3. A vector comprising: a) the nucleic acidmolecule of claim 1; and b) at least one marker gene.
 4. The vector ofclaim 3, further comprising a functional Corynebacterium replicationorigin.
 5. A method for producing a recombinant cell comprisingintroducing the vector of claim 3 into a host cell.
 6. A recombinantcell comprising the vector of claim
 3. 7. A method for replacement of awild-type pyruvate carboxylase gene, with a feedback resistant pyruvatecarboxylase gene, in a Corynebacterium glutamicum host cell comprisingthe steps of: a) replacing a genomic copy of said wild-type pyruvatecarboxylase gene with a selectable marker gene through homologousrecombination to form a first recombinant strain; and b) replacing saidselectable marker gene of step (a) in said first recombinant strain,with said feedback resistant pyruvate carboxylase gene throughhomologous recombination to form a second recombinant strain; whereinsaid homologous recombination in step (a) and (b) occurs between saidhost cell and the vector of claim
 3. 8. A recombinant strain produced bythe method of claim
 7. 9. An isolated or purified nucleic acid moleculecomprising a nucleic acid sequence encoding a pyruvate carboxylaseenzyme desensitized to feedback inhibition by aspartic acid, said enzymehaving an amino acid sequence that differs from SEQ ID NO: 19 by atleast one but no more than six mutations, said at least one, but no morethan six mutations selected from the group consisting of: a) glutamicacid at position 153 is replaced with an aspartic acid, b) alanine atposition 182 is replaced with a serine, c) alanine at position 206 isreplaced with a serine, d) histidine at position 227 is replaced with anarginine, e) alanine at position 455 is replaced with a glycine, and f)aspartic acid at position 1120 is replaced with a glutamic acid.
 10. Anisolated or purified nucleic acid molecule comprising a nucleotidesequence at least 95% identical to SEQ ID NO:1 and which codes for apyruvate carboxylase enzyme desensitized to feedback inhibition byaspartic acid, wherein said pyruvate carboxylase mutations to SEQ IDNO:19 include: a) glutamic acid at position 153 is replaced with anaspartic acid, b) alanine at position 182 is replaced with a serine, c)alanine at position 206 is replaced with a serine, d) histidine atposition 227 is replaced with an arginine, e) alanine at position 455 isreplaced with a glycine, and f) aspartic acid at position 1120 isreplaced with a glutamic acid.
 11. A vector comprising: (a) the nucleicacid molecule of claim 9 or 10; and (b) at least one marker gene. 12.The vector of claim 11, further comprising a functional Corynebacteriumreplication origin.
 13. A method for producing a recombinant cellcomprising introducing the vector of claim 11 into a host cell.
 14. Arecombinant cell comprising the vector of claim
 11. 15. A method forreplacement of a wild-type pyruvate carboxylase gene, with a feedbackresistant pyruvate carboxylase gene, in a Corynebacterium glutamicumhost cell comprising the steps of: (a) replacing a genomic copy of saidwild-type pyruvate carboxylase gene with a selectable marker genethrough homologous recombination to form a first recombinant strain; and(b) replacing said selectable marker gene of step (a) in said firstrecombinant strain, with said feedback resistant pyruvate carboxylasegene through homologous recombination to form a second recombinantstrain; wherein said homologous recombination in step (a) and (b) occursbetween said host cell and the vector of claim
 11. 16. A recombinantstrain produced by the method of claim 15.